Current methods of monitoring food products for microorganisms are based on End Product Analysis and Hazard Analysis Critical Control Points (“HACCP”); however, such methods rely upon limited sampling, periodic observation of products moving along the supply chain, and lengthy analytical laboratory testing which often requires amplification of microorganisms before conducting the actual testing. Bacterial plating can take up to four days to yield results, requires extensive training and experience, and may lead to false positives. Immunoassays (e.g., ELISA) and genetic analyses (e.g., PCR) involve pre-incubation periods, lengthy processing times, costly equipment, trained personnel, and may lead to false positives due to the inability to distinguish between live and dead pathogens (Leonard et al., 2003).
To overcome such weaknesses, various technologies have since been developed, including digital PCR techniques which remain unproven (Rothrock et al., 2013); antibody-based assays which are expensive and require trained personnel (Skottrup et al., 2008); and nano-based technologies (Driskell et al., 2009). However, these methods rely on punctual sampling through HACCP practices and thus do not provide real-time monitoring.
Bacteriophages (“phages”) are highly specific, nanometer-scale bacterial viruses that exist naturally in food. Phages are commonly found in meat products at concentrations as high as 108/g (Abedon, 2008). Phage-based products have been approved as antibacterial agents to treat meat products in Europe and the United States (Housby et al., 2009). Phages require a host organism to reproduce. Their high specificity has stimulated investigation into their use in biosensors or diagnostic tools. One promising approach is to genetically modify a phage to express a reporter protein upon recognition and infection of the bacterial host; for example, reporter phages have been developed for the expression of luciferase which emits a bioluminescent signal or green fluorescent protein ((Koeris et al., 2015; Loessner et al., 1996; Rosenbloom et al., 2014; Waddell et al., 2000; Funatsu et al., 2002). Genetically modified phages have also been used in the Bacterial Ice Nucleation Diagnostic test for Salmonellae (Jay et al., 2005). Pathogens can thus be detected at concentrations as low as 10 cells/g in meat and cheese within twenty-four hours, demonstrating a significant time improvement over plaque assays, and feasibility for use in diagnostic applications (Gervais et al., 2007). However, these methods rely upon off-line sampling and suspension of phages in solutions. Immobilized phages have been used for pathogen recognition in biosensors but these have limitations including, for example, detection limits, power supply requirements, sampling limitations, and loss of signal upon host lysis (Byrne et al., 2003; Tawil et al., 2013).
Several inexpensive commercial sensors can be utilized to monitor the environmental conditions of consumer products during shipment including, for example, time-temperature indicators which change color in response to elevated temperatures (e.g., OnVu™ from BASF, Monitor Mark™ from 3M), and colorimetric systems indicative of oxygen exposure (e.g., Ageless Eye™ , Oxysense™ ). Colorimetric systems for monitoring fish freshness involve basic amines generated as fish spoils in the headspace reacting with a pH sensitive dye embedded in a polymer matrix (Byrne et al., 2003). Colorimetric systems necessitate visual inspection by eye or reader of the transitions, requiring line-of-sight access to the indicator.